Summary/Abstract Sickle cell disease (SCD), a recessive inherited disorder caused by a point mutation in the hemoglobin chain of red blood cells (RBCs). Microvascular dysfunction is central to the pathobiology of SCD, leading to life- threatening consequences. A major consequence is occlusion of activated microvasculature with sickle RBCs leading to unpredictable and frequent episodes of acute pain called vaso-occlusive crises (VOC), frequent hospitalization and poor quality of life. Many individuals with SCD suffer chronic pain that may start during infancy and continue to increase throughout life. Opioids are the mainstay for treatment but their side-effects and fear of addiction remain a major concern. Hence, a major unmet need is to prevent and/or treat pain more effectively. VOC is associated with increased hemolysis that releases free heme. Our preliminary data reveal that administration of free heme causes hyperalgesia (pain) in transgenic sickle mice expressing human sickle hemoglobin (Hb) and in control mice expressing normal human HbA. Our preliminary data shows that heme stimulates mast-cell extracellular traps (ETs) by releasing nuclear DNA and citrullinated histones. Mast- cell activation promotes hyperalgesia in sickle mice. We hypothesize that heme-induced mast-cell activation leads to release of citrullinated histones and noxious substances and contributes to inflammation, vascular dysfunction and axonal injury leading to vasoocclusion and hyperalgesia in SCD (Schema I). Mast cells may play a causal role in VOC and chronic pain in SCD. Targeting mast cells will ameliorate VOC and pain at its source. We will test our hypothesis using a translational approach with four specific aims to establish whether, heme contributes to chronic and/or acute hyperalgesia (Aim1), heme contributes to chronic/acute pain via mast-cell activation (Aim2), and heme-induced hyperalgesia is driven by novel mast cell?dependent mechanisms leading to axonal and vascular injury (Aim3), including, release of inflammatory cytokines, proteases, ETs with DNA and citrullinated histones from mast cells that cause axonal injury in the periphery and DRG neurons, and endothelial activation via endoplasmic reticulum stress. Aim4 will entail determining whether targeting the mechanisms of heme-induced mast- cell activation attenuates hyperalgesia and vaso-occlusion. We will use genetic and pharmacological approaches, namely [i] humanized transgenic HbSS-BERK sickle mice exclusively expressing human sickle Hb, [ii] HbAA-BERK control mice expressing normal human HbA, [iii] sickle mice deleted for, [a] mast cells or [b] TLR4 and their congenic controls; and mechanism-specific pharmacological inhibitors to prevent vaso- occlusion and pain. Mouse models and biologicals are available in our laboratories. By using multiple strategies in vivo and in vitro, involving mast cell?mediated hyperalgesia and their targeting with novel and/or FDA-approved drugs, we expect that our observations will lead to translationally relevant functional outcomes?reduction of VOC and acute as well as chronic pain in SCD.